Osteoblasts are thought to differentiate from pluripotent mesenchymal stem cells. The maturation of an osteoblast results in the secretion of an extracellular matrix which can mineralize and form bone. The regulation of this complex process is not well understood but is thought to involve a group of signaling glycoproteins known as bone morphogenetic proteins (BMPs). These proteins have been shown to be involved with embryonic dorsal-ventral patterning, limb bud development, and fracture repair in adult animals. B. L. Hogan, Genes & Develop., 10:1580 (1996). This group of transforming growth factor-beta superfamily secreted proteins has a spectrum of activities in a variety of cell types at different stages of differentiation; differences in physiological activity between these closely related molecules have not been clarified. D. M. Kingsley, Trends Genet., 10:16 (1994).
In addition to extracellular signals, such as the BMPs, intracellular signals or regulatory molecules may also play a role in the cascade of events leading to formation of new bone. One broad class of intracellular regulatory molecules is the LIM proteins, which are so named because they possess a characteristic structural motif known as the LIM domain. The LIM domain is a cysteine-rich structural motif composed of two special zinc fingers that are joined by a 2-amino acid spacer. Some proteins have only LIM domains, while others contain a variety of additional functional domains. LIM proteins form a diverse group, which includes transcription factors and cytoskeletal proteins. The primary role of LIM domains appears to be in mediating protein-protein interactions, through the formation of dimers with identical or different LIM domains, or by binding distinct proteins.
Applicants have previously cloned, sequenced and deduced the amino acid sequence of a human protein, named human LMP-1. The human protein demonstrates enhanced efficacy of bone mineralization in vitro and in vivo. LMP-1 contains an N-terminal PDZ domain and three C-terminal LIM domains. Applicants have also characterized several isoforms of the LMP protein: LMP-1, as discussed above, LMP-2 (which contains a 119 base pair deletion between bp 325 and 444, and a 17 bp insertion at bp 444, compared to LMP-1), LMP-3(which does not have a deletion but has a 17 bp insertion at bp 444, thus resulting in a shift in a reading frame and a stop codon at bp 505), and truncated (short) version of LMP-1, termed HLMP-1s, containing the N-terminal 223 amino acids of the full length hLMP-1, while maintaining osteoinductive activity. Liu et al, J. Bone Miner. Res; 17(3): 406-414 (2002), incorporated herein by reference in its entirety.
This short version resulted from a point mutation in one source of a cDNA clone, providing a stop codon which truncated the protein. See U.S. Pat. No. 6,300,127 (Hair), incorporated herein by reference in its entirety. The short version (LMP-1s, also known as LMP-1t or LMP-1(t)) is fully functional when expressed in cell culture and in vivo. In the invention instantly described, inventors have assessed whether a truncated form of human LMP-1 [hLMP-1(t)], lacking the three C-terminal LIM domains, triggers differentiation of pleuripotent myoblastic cells to the osteoblast lineage. It has also been reported that LMP1, LMP-3, and LMP-1t, but not LMP-2, are capable of inducing osteogenic differentiation in non-osseous cells. Accordingly, a 45 amino acid long osteogenic region of LMP1, LMP-3, and LMP-1t was identified. Liu et al (2002).
Even though the precise mechanism of LMP-1 is under investigation, it is generally thought that exogenous BMPs induce bone formation by activating Smad1 and Smad5 proteins. These proteins are targeted for degradation by Smurf1. The LMP-1 protein competes with Smad1, Smad5, and Smad6 proteins for Smurf1 binding thus increasing cellular responsiveness to exogenous BMPs. Sandagala et al., J. Biol. Chem. 281(25): 17212-17219 (2006), incorporated herein by reference in its entirety.
Previously, the inventors reported that the osteogenic region of LMP1, LMP-3, and LMP-1t proteins contains two possible candidate sites for interaction with Smurf1, or, more specifically, with a WW-2 motif of Smurf1.
Accordingly, agents which increase binding between the WW-2 motif of Smurf1 and the osteogenic region of the LMP protein will likely cause a decreased ubiquitination of Smad proteins and thus make the Smad proteins more available for the osteogenic signaling cascade caused by BMP. Similarly, agents which disrupt the binding between the WW-2 motif of Smurf1 and the osteogenic region of the LMP protein will likely cause an increased ubiquitination of Smad proteins and thus make the Smad proteins less available for the osteogenic signaling cascade caused by BMP.
Currently, the use of BMPs is feasible for many patients with bone healing needs due to an unexpectedly high dose which is required in humans, which results in a very high cost of BMP therapy. A 15,000 fold higher concentration of BMP-2 is required to induce bone healing in humans (1.5 mg/mL) than in cell culture (100 ng/mL). Thus, there is a need for identification of agents which can affect the osteogenic effect of BMP.